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Diabetes 51:2709-2718, 2002
© 2002 by the American Diabetes Association, Inc.

Expression of Connective Tissue Growth Factor Is Increased in Injured Myocardium Associated With Protein Kinase C ß2 Activation and Diabetes

Kerrie J. Way1, Keiji Isshiki1, Kiyoshi Suzuma1, Tamotsu Yokota1, Dmitriy Zvagelsky2, Frederick J. Schoen2, George E. Sandusky3, Penelope A. Pechous3, Chris J. Vlahos3, Hisao Wakasaki1, and George L. King1

1 Research Division, Joslin Diabetes Center, Boston, Massachusetts
2 Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts
3 Cardiovascular Research, Lilly Research Laboratories, Indianapolis, Indiana


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Protein kinase C (PKC) ß isoform activity is increased in myocardium of diabetic rodents and heart failure patients. Transgenic mice overexpressing PKCß2 (PKCß2Tg) in the myocardium exhibit cardiomyopathy and cardiac fibrosis. In this study, we characterized the expression of connective tissue growth factor (CTGF) and transforming growth factor ß (TGFß) with the development of fibrosis in heart from PKCß2Tg mice at 4–16 weeks of age. Heart-to-body weight ratios of transgenic mice increased at 8 and 12 weeks, indicating hypertrophy, and ratios did not differ at 16 weeks. Collagen VI and fibronectin mRNA expression increased in PKCß2Tg hearts at 4–12 weeks. Histological examination revealed myocyte hypertrophy and fibrosis in 4- to 16-week PKCß2Tg hearts. CTGF expression increased in PKCß2Tg hearts at all ages, whereas TGFß increased only at 8 and 12 weeks. In 8-week diabetic mouse heart, CTGF and TGFß expression increased two- and fourfold, respectively. Similarly, CTGF expression increased in rat hearts at 2–8 weeks of diabetes. This is the first report of increased CTGF expression in myocardium of diabetic rodents suggesting that cardiac injury associated with PKCß2 activation, diabetes, or heart failure is marked by increased CTGF expression. CTGF could act independently or together with other cytokines to induce cardiac fibrosis and dysfunction.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cardiomyopathy associated with diabetes can occur regardless of coronary artery disease and is characterized by myocyte hypertrophy and fibrosis (1,2). Ventricles from diabetic patients show accumulation of PAS-positive glycoproteins, collagen, and active fibroblasts (2). Similarly, ventricle from diabetic rodents show increased fibrosis and expression of the extracellular matrix (ECM) components fibronectin, collagen IV, and collagen VI (3–5). Ventricular remodeling along with changes such as defective calcium transport or fatty acid metabolism can impair contractility. Animals and patients with diabetes show reduced stroke volume and increased end-diastolic pressure (6–9), which collectively could precipitate heart failure.

Activation of the protein kinase C (PKC)/diacylglycerol (DAG) signaling pathway is one mechanism by which hyperglycemia may exert adverse cardiovascular effects. Inoguchi et al. (10) reported increased membranous PKC activity and total DAG in diabetic rat heart. PKCß2 isoform was preferentially increased in the membranous fraction of heart and aorta, suggesting that this isoform contributes to diabetic vascular complications. Indeed, administration of the PKCß selective inhibitor LY333531 to diabetic rats ameliorated increased glomerular filtration, albumin excretion, and retinal circulation rates (11). Ventricles from patients with end-stage heart failure show increased expression of PKCß and increased membranous PKC activity within cardiac myocytes (12). Furthermore, targeted overexpression of PKCß2 in mouse myocardium resulted in left ventricular hypertrophy, fibrosis, and decreased left ventricular performance, similar to cardiomyopathy of diabetic or nondiabetic origin (13,14).

PKCß2 activation may contribute to myocardial hypertrophy and fibrosis by altering expression of specific cytokines. Transforming growth factor ß (TGFß) and connective tissue growth factor (CTGF) can induce production of collagen and fibronectin from cardiac fibroblasts and myocytes, and increased expression of both has been documented at sites of myocardial infarction (15,16). CTGF has a unique TGFß response element in its promoter region (17), and many reports suggest that it acts downstream of TGFß. Increased TGFß expression is reported in kidney, renal plasma, and heart from rats and patients with diabetes (18–23). To date, glucose-induced increases in CTGF expression have only been reported in rat and human mesangial cells exposed to high glucose and in glomeruli from diabetic rats (24,25). Therefore, we have correlated PKCß2 activation with the development of cardiac fibrosis and expression of CTGF and TGFß in transgenic mice overexpressing PKCß2 in the myocardium. We have also assessed cardiac expression of CTGF in rodent models of diabetes.


   RESEARCH DESIGN AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESEARCH DESIGN AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Transgenic mice overexpressing PKCß2 in the myocardium.
Construction of the adult cardiac myocyte-specific {alpha} myosin heavy chain promoter PKCß2 transgene as well as generation of transgenic mice overexpressing PKCß2 in the myocardium and their characterization are as previously described (13). In this study, heterozygous transgenic line 4 mice and nontransgenic littermate controls were used at 4, 8, 12, and 16 weeks of age. A separate group of mice were treated with the PKCß-specific inhibitor LY333531 (0.18% in diet) (Eli Lilly, Indianapolis, IN), which was administered at weaning (3 weeks) and maintained until death at 8 weeks of age.

Diabetes induction.
Diabetes was induced in 8-week-old male FVB mice (Taconic, Germantown, NY) by injection with streptozotocin (STZ; 100 mg/kg i.p.) (Sigma, St Louis, MO) on 2 consecutive days after overnight fast. A separate group of STZ-treated mice were also treated with LY333531 (0.18% in diet) for 8 weeks. Diabetes was induced in 6-week-old male Sprague-Dawley rats (Taconic) by a single STZ injection (60 mg/kg i.p.) after overnight fast. All control animals received citrate vehicle (0.01 mol/l, pH 4.5). Two groups of STZ-treated rats were established to receive either insulin pellet implant (Linplant; Linshin, Scarborough, Ontario, Canada) or LY333531 (0.062% in diet) for 4 weeks. Successful induction of diabetes was confirmed by elevated blood glucose levels (>250 mg/dl) (Glucometer Elite XL; Bayer, Elkhart, IN). Mice were killed 8 weeks after injection, and rats were killed at 2, 4, and 8 weeks. All procedures described are in accordance with the Joslin Diabetes Center’s Animal Care and Use Committee guidelines.

RNA isolation and Northern blot analysis.
Total RNA was isolated by the phenol/guanidine thiocynate/chloroform method (13). Northern blot analysis was performed using total RNA (15–25 µg) following standard techniques (26). Hybridization was performed with the following cDNA probes: human CTGF cDNA EcoRI fragment (0.8 kb) (26); mouse {alpha}1(VI) collagen cDNA EcoRI fragment (2 kb) (13); and mouse fibronectin cDNA EcoRI and XhoI fragment (mouse fibronectin clone ID 524915; American Type Culture Collection, Manassas, VA). Differences in RNA loading were normalized using a PstI fragment of human acidic ribosomal phosphoprotein cDNA (36B4) as the internal control gene, and data were expressed as fold change from control values.

Mutiplex RT-PCR.
Total RNA (500 ng) was reverse transcribed at 42°C in a 25-µl volume that included 50 ng random hexamer primers (Life Technologies, Rockville, MD) and 200 units Superscript II RT (Life Technologies). The following mouse primers were used: TGFß 5'-CAC.CTG.CAA.GAC.CAT.CGA.CAT, 3' -AAA.GAC.AGC.CAC.TCA.GGC.GTA.TC (Genebank accession no. NM-011577); TATA binding protein (TBP) 5'-ACC.CTT.CAC.CAA.TGA.CTC.CTA.TG, 3' -ATG.ATG.ACT.GCA.GCA.AAT.CGC (Genebank accession no. D01034). Signals to TGFß were normalized using TBP as the internal standard. Band identity was confirmed using Southern Blot analysis and DNA sequencing. Multiplex quantitative PCR was performed using cDNA (20 ng) in a 47-µl mix that included 1.5 mmol/l MgCl2 (Perkin Elmer, Foster City, CA), 80 µmol/l dNTP, 10 pmol oligonucleotide primer, 5 units AmpliTaq Gold DNA polymerase (Perkin Elmer), and 2.5 µCi [{alpha}-32P]dCTP. Reaction conditions commenced for 10 min at 94°C followed by 24 cycles for 1 min at 94°C, 1 min at 55°C, and 1 min at 72°C and then final incubation at 72°C. Amplimers were separated on a polyacrylamide gel for analysis.

Western blot analysis.
Ventricle samples were prepared as described (27), and 50 µg SDS extract (S2) was resolved by 4–12% SDS/PAGE. Rat heart collagen VI standard was used (provided by Dr. M.J. Spiro) (4), and membranes were incubated with collagen VI primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA).

Histological examination.
Formalin (10%) fixed hearts were bisected transversely at the mid-ventricular level and embedded in paraffin. Sections (~5 µm thick) were stained with hematoxylin and eosin as well as Masson’s trichrome stain. To determine cardiac myocyte size, the diameter (microns) of 25 myocytes was assessed under x400 magnification within the left ventricle and a mean was obtained. Fibrosis was quantitated using trichrome-stained sections. Dark blue staining collagen fibers were quantitated as a measure of fibrosis. Color images (six to eight fields per heart section) were obtained under x40 magnification and digitized using Image-Pro Plus version 3.0 software (Media Cybernetics, Silver Spring, MD). Areas of fibrosis were divided by the area of tissue section measured.

Immunostaining.
Before immunostaining, antigen retrieval was performed on sections by immersion in Target Retrieval Solution (15 min at 85°C) (Dako, Carpinteria, CA). Endogenous peroxide was blocked using 3% H2O2 (5 min). After 45 min of blocking using protein blocking solution (Dako), sections were incubated with 10 µg/ml primary antibody to PKCß2 (Santa Cruz Biotechnology) or CTGF (Cell Sciences, Norwood, MA). For immunostaining, biotinylatedlinked streptavidin-horse radish peroxidase (Dako LSAB2) antibody was used with DAB chromagen. Slides were counterstained with hematoxylin. Sections for immunofluorescence studies were double stained. CTGF was detected with anti-rabbit Alexa 488 and PKCß2 with anti-rabbit Alexa 568 (Dako).

Statistical analysis.
Statistical analyses were performed using unpaired Student’s t test and one-way ANOVA, and P values <0.05 indicated significance (SigmaStat Software; Jandel Scientific). Values shown are means ± SE, and n indicates the number of animals used.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESEARCH DESIGN AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of PKCß2 overexpression on growth factors and ECM expression.
Body weight (g) and whole heart weight (mg) were assessed from control mice and transgenic mice overexpressing PKCß2 in the myocardium at 4, 8, 12, and 16 weeks of age. No significant differences in body weights were observed between the control and transgenic groups at any age (data not shown). Heart weights from transgenic mice increased significantly (P < 0.05) at 8 and 12 weeks of age (138.4 ± 7.1 and 156.1 ± 13.2 mg, respectively) compared with the control group (109.8 ± 3.8 and 114.4 ± 4.7 mg, respectively; n = 12–22). No significant differences (P > 0.05) in heart weights were observed between the control and transgenic groups at 4 weeks (110.2 ± 5.6 and 106.4 ± 4.7 mg, n = 17–19) or 16 weeks (109.3 ± 3.4 and 111.6 ± 3.9 mg, n = 18–23). Similarly, significantly increased (P < 0.05) heart-to-body weight ratios were observed in transgenic mice at 8 weeks (5.7 ± 0.5) and 12 weeks (5.6 ± 0.4) compared with the control group (4.4 ± 0.1 and 4.3 ± 0.1, respectively). At 16 weeks, atrophy of transgenic hearts resulted in similar ratios between the control (4.1 ± 0.1) and transgenic groups (4.3 ± 0.1). A separated group of mice were treated with the PKCß-specific inhibitor LY333531 for 8 weeks. No differences in body weight, heart weight, or heart-to-body weight ratios were observed between the control (26.4 ± 1.2 g, 129.1 ± 7.0 mg, and 4.9 ± 0.1, respectively; n = 5) and transgenic groups (25.9 ± 1.0 g, 135.5 ± 7.8 mg, and 5.2 ± 0.1, respectively; n = 6).

Northern blot analysis of total RNA isolated from ventricle from transgenic mice at 4, 8, 12, and 16 weeks of age revealed a single 3.6-kb transcript for PKCß2 mRNA (data not shown). Endogenous PKCß2 mRNA levels from control ventricles at all durations were almost undetectable. Expression of collagen VI (Fig. 1A) and fibronectin (Fig. 1B) mRNA levels increased significantly (average of 1.6-fold and 2.3-fold, respectively; P < 0.05) in ventricles from transgenic mice at 4, 8, and 12 weeks of age compared with control mice. Collagen VI protein expression also increased significantly (P < 0.05) in ventricle from 8-week transgenic mice by 1.7 ± 0.2-fold compared with controls (1.0 ± 0.0-fold, n = 5) (Fig. 1C). Treatment of control and transgenic mice with LY333531 for 8 weeks reduced this increase in collagen VI protein expression in transgenic hearts to 1.3 ± 0.2-fold, which did not differ significantly (P > 0.05) from control values (1.0 ± 0.1-fold, n = 4) (Fig. 1C). However, expression of collagen VI mRNA remained elevated by 2.1 ± 0.2-fold compared with controls (1.0 ± 0.1-fold, n = 5–6). At 16 weeks, mRNA levels for collagen VI and fibronectin did not differ significantly (P > 0.05) between the groups (Fig. 1A and B). In contrast, collagen VI protein expression in 16-week transgenic hearts revealed a 1.4-fold (n = 2) increase in expression compared with the control group (Fig. 1C).



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  		FIG. 1. Effect of PKCß2 overexpression in the myocardium on collagen VI (CoVI) and fibronectin (Fn) expression in the heart. Northern blot images of CoVI (A), Fn (B), and 36B4 mRNA (A and B) obtained from control (C) and transgenic mice (T) at 4, 8, 12, and 16 weeks of age are shown. Quantification of CoVI or Fn mRNA normalized to 36B4 from control ({square}) and transgenic mice ({blacksquare}) at different ages are plotted. C: Representative immunoblots showing collagen VI protein expression in heart from 8- and 16-week control (C) and transgenic mice (T). A separate group of control and transgenic mice were treated with the PKCß inhibitor LY333531 for 8 weeks. Protein (50 µg) from whole ventricle showed reactivity to collagen type VI, and the 150-kDa band was confirmed by using a prestained molecular weight marker (not shown) and by comparison with a rat heart collagen VI standard (Std). Results are means ± SE of four to eight experiments. *P < 0.05 vs. corresponding control value (unpaired t test).

 
Significant increases (P < 0.05) in CTGF mRNA expression, from 2.5- to 3.3-fold, were observed in ventricle obtained from transgenic mice at 4, 8, and 12 weeks duration compared with the control group (Fig. 2A). At 16 weeks, a 1.5-fold increase in CTGF expression was observed and remained significantly greater (P < 0.05) than the control group. At 4 weeks duration, TGFß mRNA levels did not differ between groups, and at 8 and 12 weeks, significant increases (P < 0.05) in TGFß expression between 1.4- and 1.9-fold were observed compared with controls (Fig. 2B). However, after 16 weeks, no difference in TGFß expression between the groups was apparent.



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  		FIG. 2. Effect of PKCß2 overexpression in the myocardium on CTGF and TGFß mRNA expression in the heart. Northern blot images of CTGF and 36B4 mRNA (A) and TGFß and TBP mRNA (B) obtained from control (C) and transgenic mice (T) at 4, 8, 12, and 16 weeks of age are shown. Multiplex PCR was used to quantitate expression of TGFß mRNA, and confirmational experiments were performed (data not shown) to verify the validity of the amplimers and to ensure linearity of the reaction conditions. Quantification of CTGF or TGFß mRNA normalized to the appropriate control gene from control ({square}) and transgenic mice ({blacksquare}) at different ages are plotted. The effect of LY333531treatment for 8 weeks on the expression of CTGF (C) and TGFß (D) from control and transgenic mice are also shown. Results are means ± SE from 3 to 17 experiments. *P < 0.05 vs. corresponding control value (unpaired t test).

 
Treatment of control and transgenic mice with LY333531 for 8 weeks reduced the increase in expression of CTGF from 3.3-fold in untreated transgenic mice to 1.7 ± 0.3-fold in transgenic mice treated with LY333531, which did not differ significantly from controls treated with LY333531 (1.0 ± 0.2-fold) (Fig. 2C). Interpretation of the effect of LY333531 treatment on TGFß expression is confusing (Fig. 2D). Expression of TGFß did not differ significantly between control and transgenic mice treated with LY333531 (Fig. 2D). LY333531 induced a slight reduction in expression of TGFß, from 1.4 ± 0.1-fold in the untreated transgenic group to 1.3 ± 0.1-fold in the treated group, which did not differ from the control group treated with LY333531 (1.0 ± 0.1-fold). We interpret these findings to indicate that LY333531 did not affect expression of TGFß in transgenic hearts compared with controls.

Effect of PKCß2 overexpression on cardiac histology.
Ventricular myocardium from 4- to 16-week control mice had normal histology (Fig. 3A). In contrast, ventricular sections from transgenic mice at all time points displayed myocyte hypertrophy with focal myofiber disarray (Fig. 3B). In 4-week transgenic hearts (Fig. 3C), sites of dystrophic calcification associated with inflammatory cell infiltrate and necrosis commonly occurred. In addition, patchy fibrosis was noted in transgenic hearts at all durations (Fig. 3D and E). Myocardial pathology evolved from 8 to 16 weeks with a reduction in cell infiltration and progressive scar maturity by 16 weeks (Fig. 3F). Increased cardiac myocyte size, indicating myocyte hypertrophy, was determined in ventricles from transgenic mice at all durations and reached significance at 4, 12, and 16 weeks of age (P < 0.05) (Fig. 4A). Quantitation of the area of fibrosis/area measured (percent) confirmed that there was a significant increase in sections from transgenic mice at all durations (P < 0.05) (Fig. 4B) compared with controls. In all sections from transgenic mice quantitated, 2–3% of the measured area was shown to be fibrotic, whereas <0.1% of ventricle sections from control mice exhibited fibrosis.



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  		FIG. 3. Photomicrographs of ventricular sections obtained from control mice and transgenic mice overexpressing PKCß2 in the myocardium. Hematoxylin and eosin staining (A–D), demonstrating normal cardiac histology from a 16-week control heart (A). Arrows indicate cardiac myocyte nuclei. Ventricular sections from transgenic mice had myofiber disarray (B; 16 weeks) and a heterogeneous population of cardiac myocytes with complicated nuclei (arrows), indicating cardiac myocyte hypertrophy. Patches of dystrophic calcification with mononuclear inflammatory infiltrate (C; 4 weeks) and sites of fibrosis (D; 8 weeks) were observed. Masson’s trichrome staining (E and F) demonstrated the progression of inflammatory cell infiltration with deposition of collagen at 4 weeks (E) to the formation of a dense collagenous scar by 16 weeks (F). (magnification: A, B, and D–F: x400; C: x200).

 


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  		FIG. 4. Effect of PKCß2 overexpression in the myocardium on cardiac myocyte size (A) and area of fibrosis (B) in heart. Myocyte size (microns) and area of fibrosis (shown as percent and normalized to area measured) were determined from histological sections of heart obtained from control ({square}) and transgenic mice ({blacksquare}) overexpressing PKCß2 in the myocardium at 4, 8, 12, and 16 weeks of age. Results are means ± SE from three animals. *P < 0.05 vs. corresponding control value (unpaired t test).

 
Immunostaining of PKCß2 and CTGF in ventricle from mice overexpressing PKCß2 in the myocardium.
Ventricular sections from 8-week transgenic mice displayed patchy or scattered immunostaining for PKCß2. Intense staining was observed in some cardiac myocytes, with adjacent myocytes staining weakly (Fig. 5A). In sections from control mice, staining for PKCß2 could not be detected (Fig. 5B). Immunofluorescence was used to assess for colocalization of PKCß2 and CTGF within cardiac myocytes. A similar pattern of patchy staining for PKCß2 to cardiac myocytes was observed in transgenic sections after immunofluorescent staining (Fig. 5C). Immunofluorescent staining for CTGF was also observed in cardiac myocytes from transgenic hearts (Fig. 5D); staining was weaker compared with that for PKCß2, and some bleed-over fluorescence could be seen. Using double-labeled immunofluorescence, colocalization of PKCß2 and CTGF in cardiac myocytes was also observed in some cells (Fig. 5E). To further clarify the colocalization, the double-stained slides were examined using confocal microscopy to eliminate the fluorescent bleed over between the wavelengths used. The confocal images revealed colocalization similar to that seen with epifluorescence (Fig. 5F).



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  		FIG. 5. Photomicrographs of ventricular sections from 8-week control mice (B) and transgenic mice overexpressing PKCß2 in the myocardium (A and C–F). Immunostaining for PKCß2 localized to cardiac myocytes is shown in patches within transgenic sections (A; arrows). Staining for PKCß2 could not be detected in control sections (B). A similar pattern of staining for PKCß2 was observed in transgenic sections after immunofluorescent staining (C; Alexa 568, arrows). Immunofluorescent staining for CTGF was also observed in cardiac myocytes from transgenic hearts (D; Alexa 488, arrows). Staining for CTGF appeared weaker than that for PKCß2 and some bleed over of fluorescence was observed (D). Colocalization of PKCß2 and CTGF in some cardiac myocytes is shown (E; bright yellow, arrows). Specific immunostaining of these factors was not observed in sections from control mice (data not shown). Confocal images of PKCß2 and CTGF further confirmed colocalization in cardiac myocytes from transgenic hearts (F; yellow staining, arrows). Immunostained and fluorescent slides were viewed with a standard Leitz using a filter to detect 488/568-nm wavelengths, and the slides were reexamined on a a Bio-Rad MRC1024-UV confocal microscope (Bio-Rad, Hernel, U.K.) using a 40x Plan Apo 1.3-oil immersion objective on a Nikon Diapot 200 inverted microscope. The red and green fluorescent images were collected sequentially with Lasersharp 3.1 acquistion software after the background fluorescence of a no primary antibody control slide was set below the threshold of detection. Images were collected as a stack in the .PIC format to be analyzed for colocalization of PKCß2 and CTGF (magnification: A–E: x200; F: x650).

 
Effect of diabetes on cardiac expression of CTGF.
The effect of diabetes on CTGF mRNA expression in ventricle was assessed in 8-week diabetic mice and in 2-, 4-, and 8-week diabetic rats. At all durations, blood glucose levels of STZ-treated mice and rats were significantly elevated (P < 0.05) compared with the corresponding vehicle-treated group (Table 1). Insulin treatment of 4-week diabetic rats reduced the increase in blood glucose levels, whereas for diabetic mice or rats treated with LY333531, levels remained significantly greater (P < 0.05) than the control group. Increases in body weight of STZ-treated mice and rats at all durations tested were significantly reduced (P < 0.05) compared with vehicle-treated animals, although a steady increase in body weight was observed in both the control and diabetic groups (Table 1). Insulin treatment of diabetic rats prevented body weight loss, whereas LY333531 did not affect the body weight of diabetic animals.


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  		TABLE 1 Blood glucose levels and body weights of vehicle- and STZ-treated mice and rats at different time periods after injection

 
Ventricle from 8-week diabetic mice showed a significant 2.4-fold increase (P < 0.05) in CTGF mRNA expression compared with the control group (Fig. 6A). Assessment of collagen VI or fibronectin mRNA levels revealed no difference between 8-week control and diabetic mice (data not shown). Treatment of diabetic mice with LY333531 for 8 weeks did not affect the increase in CTGF mRNA expression compared with controls. TGFß mRNA expression was also significantly increased, by 3.5-fold, in diabetic mice compared with controls (Fig. 6B). Treatment of diabetic mice with LY333531 for 8 weeks prevented the increase in TGFß mRNA expression compared with the control group (Fig. 6B).



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  		FIG. 6. Effect of diabetes on CTGF and TGFß mRNA expression in mouse heart. Northern blot images of CTGF and 36B4 mRNA (A) and TGFß and TBP mRNA (B) from 8-week control and diabetic mice and diabetic mice treated with LY333531. Quantification of CTGF or TGFß mRNA normalized to the appropriate control gene from control ({square}) and diabetic mice ({blacksquare}) are plotted. Results are means ± SE from five to eight experiments. * P < 0.05 vs. corresponding control value (one-way ANOVA).

 
Ventricle from 2-, 4-, and 8-week diabetic rats showed significant increases (P < 0.05) in CTGF expression of 1.3-, 1.3-, and 1.5-fold, respectively, compared with controls (Fig. 7A and B). Expression of collagen VI mRNA in heart from 4-week diabetic rats did not differ from the control group (data not shown). Treatment of 4-week diabetic rats with insulin prevented the increase in CTGF mRNA expression (Fig. 7C), whereas treatment with LY333531 had little effect on CTGF expression such that levels did not differ significantly from either the untreated diabetic or control groups (Fig. 7C).



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  		FIG. 7. Effect of diabetes on CTGF expression in rat heart. A: Northern blot images of CTGF and 36B4 mRNA obtained from control and diabetic rats 2, 4, and 8 weeks after treatment. B: Quantification of CTGF mRNA normalized to 36B4 from control ({square}) and diabetic rats ({blacksquare}) with increasing diabetes duration. Quantification of CTGF mRNA expression in ventricle from 4-week diabetic rats treated with insulin and LY333531 (C). Results are means ± SE from 4–11 experiments. *P < 0.05 vs. corresponding control value (unpaired t test and one-way ANOVA).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have characterized the effect of PKCß2 isoform activation on the expression of CTGF, TGFß, and ECM proteins and on pathological fibrosis using PKCß2 transgenic mice and have identified the potential for similar alterations in rodent models of diabetes. Preferential activation of PKCß2 isoform was identified in diabetic rat heart (10), which leads to the generation of transgenic mice with sustained cardiac activation of PKCß2 as an accelerated cardiac model of later-stage diabetes. We have reported that ventricle from these mice developed hypertrophy, fibrosis, and contractile dysfunction at 8–12 weeks of age (13). Studies by Bowman et al. (14), in which PKCß was constitutively activated in adult mouse heart after tetracycline induction, noted progressive cardiac hypertrophy and dysfunction, although no fibrosis by 9 months. Our study focused on the changes in cytokines, ECM, and fibrosis in transgenic mice and diabetic rodents in order to determine a relationship between PKCß isoform activation and CTGF with fibrosis because such pathologies are observed in cardiomyopathy associated with diabetes and heart failure.

In PKCß2 transgenic mice, gross pathological changes and increased heart weight were not observed until 8 weeks of age, although increased PKCß2 expression was observed from 2 to 3 weeks of age postpartum (13). In addition, immunoblot analysis of membranous heart fraction confirmed no change in activity of other PKC isoforms, including PKC{alpha}, -{delta}, or -{varepsilon} (13). Histological abnormalities, such as fibrosis and myocyte hypertrophy, occurred at 4 weeks and probably were present at 1–2 weeks of age. Cardiac myocyte size was increased at all durations, whereas heart weight increased significantly at 8 and 12 weeks of age. By 16 weeks, atrophy or normalization of heart weight occurs, although cardiac myocyte hypertrophy was still apparent at this duration. At 16 weeks, heart weight may also be influenced by contraction of replacement fibrosis of injured myocytes and by a decrease in new fibrotic sites resulting from loss of myocytes overexpressing PKCß2. It is interesting that the early lesions manifested as patchy areas of leukocyte infiltrate, calcium deposits, and myofiber disarray with occasional myocyte lysis, indicating an inflammatory response to injury. The exhibition of focalized lesions suggested that the expression of PKCß2 in the myocardium is not homogenous, and immunohistochemical results demonstrating patchy staining for PKCß2 to cardiac myocytes support this. This is unexpected because the {alpha} myosin heavy chain promoter was used for targeting PKCß2 isoform expression and suggests that the location of transgene incorporation in the chromosome may influence its expression.

Activation of the PKCß isoform clearly induced cardiomyopathy with hypertrophy of myocytes and fibrosis. The presence of fibrosis was further supported by the quantitation of mRNA levels of collagen type VI and fibronectin. Increased expression of these proteins in 4- to 12-week transgenic mice coincided with the increase in cardiac myocyte size and the histological appearance of fibrosis; however, by 16 weeks, expression did not differ from control levels. Normalization in ECM expression occurs even though elevated PKCß2 expression persists. However, both histological analysis and immunoblot assessment showed increased fibrosis and collagen VI protein in heart from 8-week transgenic mice, which continued until 16 weeks, indicating that pathology was still present at this latter duration. An association of PKCß with these pathologies was confirmed using the PKCß selective inhibitor LY333531, which reduced the increase in heart weight and collagen VI protein expression and, as previously reported, reduced the size of fibrotic lesions (13). We believe that LY333531 acts to reduce rather than prevent potential cardiac alterations, as it is administered to 3-week-old weaned mice in which myocardial changes may be initiated. It is unclear why the increase in collagen VI mRNA was not reduced by drug treatment; it is possible that transcript half-life or stability is altered or that continued drug treatment may be necessary to reduce levels. Therefore, initial PKCß2 activation parallels the induction of a fibrotic response that may involve stimulation of cytokine expression, and this coincides with myocyte injury and hypertrophy. Indeed, an increase in collagens and fibronectin is linked to cardiac failure, hypertrophy, infarction, and diabetes, and collagens and fibronectin also exhibit activation or increased expression of the PKCß isoform (2–5,22,28–30).

Our investigation of cytokines, which can induce the fibrotic response, revealed two potential growth factors: TGFß, which is known to be regulated by PKC, and CTGF, which has not been linked to PKC; however, both factors possess AP-1 binding sites in their promoter regions. TGFß is induced in cardiac pathologies, including fibrosis, hypertrophy, and infarction, and precedes elevation of ECM proteins (16,30–33). Recently, the time-course for increased TGFß expression after myocardial infarction was associated with increased expression of CTGF and was attributed to a TGFß response element in the CTGF promoter region (16,17). Both correlated with increased fibronectin and collagen expression.

Since its identification and classification, CTGF has been associated with fibrotic disorders of the pancreas, skin, lung, liver, and atherosclerotic plaques (34–40). Moreover, addition of TGFß increases CTGF mRNA in cardiac fibroblasts, rat neonatal cardiac myocytes, and human cardiac fibroblasts (16). Reports have also shown a lack of dependence among these cytokines. For example, dexamethasone increases CTGF expression and downregulates TGFß in Balb 3T3 cells (41); increased CTGF and collagen expression in the absence of a TGFß transcript was reported in pseudoscleroderma associated with lung cancer (42); and CTGF can autoinduce CTGF expression in mesangial cells without affecting TGFß levels (25).

Alterations in cardiac CTGF expression were of particular interest given recent reports indicating its role in myocardial infarction and fibrosis, elevated expression in heart from ischemic patients (15,16), and localization to cardiac myocytes, fibroblasts, and myoblasts and sites of fibrotic lesions in rodent heart (15). In our study, increased expression of TGFß in transgenic hearts was observed at 8 and 12 weeks of age, corresponding to increased heart weight. At 16 weeks, no difference in TGFß expression between the control and transgenic groups was observed. In contrast, significant increases in CTGF expression occurred at all durations, demonstrating that increased expression of CTGF coincided with increased expression of PKCß2, collagen VI, and fibronectin, before the elevation of TGFß, and persisted even when TGFß levels normalized at 16 weeks. Therefore, in this model, cardiac CTGF expression can be modulated independently of TGFß and may contribute to elevated ECM gene expression and fibrosis. The reduced expression of CTGF in transgenic mice treated with LY333531 indicates increased PKCß expression associates with increased CTGF expression and myocardial injury. To further confirm an association between PKCß2 and CTGF, immunohistochemical localization of CTGF to cardiac myocytes colocalized in some cells with staining for PKCß2. LY333531 did not appear to affect TGFß expression; expression remained elevated, but the difference was not significant. A longer treatment period may be effective in reducing levels further. Therefore, increased CTGF gene expression associated with PKCß2 activation occurs early, indicating a potential role in inducing fibrotic change; furthermore, CTGF expression is sustained longer than TGFß, indicating potential for a role in chronic fibrosis.

Whether elevated CTGF expression alone is sufficient to induce fibrotic changes in this model or an associated increase in TGFß is required is unknown. Further studies are required to more definitely assess the association between these cytokines and the production of cardiac fibrosis, although previous studies indicate a strong link between increased TGFß and CTGF expression and increased ECM components in cardiac and renal tissue (15,16,24, 25). Furthermore, although a parallel exists between increased PKCß2, CTGF, and TGFß expression with myocardial injury in transgenic mouse heart, it remains to be determined whether PKCß2 activation can directly induce these cytokines or whether the increase is secondary to PKCß2-induced cardiac cytoxicity, including necrosis and apoptosis.

The effect of diabetes/high glucose on CTGF expression has been assessed in renal tissue only. Mesangial cells exposed to high glucose show increased expression of CTGF, which could be reduced with an anti-TGFß antibody and by PKC inhibition, indicating that both TGFß-dependent and -independent mechanisms were involved (24, 25). In addition, increased CTGF expression was reported in diabetic rat and mouse renal cortex and glomeruli (24,25). We now report for the first time that CTGF expression is increased in ventricle from diabetic mouse and rat heart and that insulin can prevent this increase. In heart from diabetic mice, a corresponding increase in TGFß expression was noted; similar diabetes-induced increases in TGFß have been reported in the diabetic kidney and heart, and it has been determined that its promoter region possesses a glucose response element (18–23). Unlike heart from our transgenic model, which showed a fivefold increase in PKC activity (13), we did not identify increased expression of collagen VI or fibronectin mRNA in heart from diabetic rodents. We have previously reported that hearts from 2-week diabetic rats show a 21% increase in membranous PKC activity that is significantly less than that in the transgenic model. Longer durations of diabetes than those used in this study may be necessary to see pathological alterations comparable to those observed in the transgenic mice. For instance, 5-month diabetic rats were used to identify increased cardiac fibrosis, apoptosis, and dysfunction (43). To determine an association between increased cardiac PKCß expression in the diabetic heart with cytokine expression, animals were treated with LY333531. Inhibition of PKCß prevented the increase in cardiac expression of TGFß; however, CTGF expression remained increased in the diabetic rat and mouse heart, indicating a lack of association between PKCß and increased CTGF expression. These findings could indicate that longer durations of treatment with LY333531 may be necessary to see further reductions in expression of CTGF or that other isoforms of PKC in the diabetic heart are associated with cardiac pathologies. PKC isoforms, including PKC{alpha}, -{delta}, -{varepsilon}, and -{zeta}, have been reported to show increased membranous expression in diabetic heart (44–47). In addition, roles for PKC{alpha} and -{varepsilon} have been identified in cardiac hypertrophy and heart failure, and it was reported that different levels of PKC{varepsilon} activation could induce dichotomous cardiac phenotypes (12,48–50). Furthermore, the findings could indicate that other diabetes-associated mechanisms unrelated to PKC activation may be involved. Nevertheless, an association between increased PKCß and TGFß expression is indicated in diabetic heart, whereas hyperglycemia is associated with increased cardiac CTGF expression. Future long-term diabetes studies will help to determine the interdependence of these cytokines and their contribution to cardiac pathologies.

In this study, we have shown that PKCß2 activation is associated with increased myocardial injury and increased expression of the cytokines CTGF and TGFß. It remains to be determined whether PKCß2 activity induces cytokine expression to then produce cardiac injury, such as fibrosis and hypertrophy, or whether PKCß2 is directly toxic to the myocardium, leading to increased cytokine expression. We report for the first time that in diabetic heart, CTGF expression is increased and may play a role in associated pathologies. Although the mechanisms involved in contributing to the diabetes-induced increase in CTGF expression remain unclear, an increase in cardiac CTGF expression may be a marker of myocardial injury. Given that increased cardiac fibrosis ultimately leads to ventricular stiffness regardless of cardiac hypertrophy, therapies aimed to reduced ECM accumulation are required. We now provide evidence to suggest that inhibition of PKCß activation or the expression of cytokines, such as CTGF, could prevent cardiac pathologies associated with ECM accumulation, particularly in diabetes or heart failure.


   ACKNOWLEDGMENTS
 
This study was supported by the grants NIH EY5110, JDF 22380811, and NIH DK59725-01. K.J.W. is the recipient of a JDFI Postdoctoral Fellowship.

The authors thank Dr. Myra Lipes and Edward Boschetti from the Transgenic Core at the Joslin Diabetes Center for the generation of transgenic mice. They also appreciate the technical assistance provided by Helen Shing, Darren Opland, and Ravi Rauniyar.


   FOOTNOTES
 
Address correspondence and reprint requests to George L. King, MD, Harvard Medical School, Joslin Diabetes Center, One Joslin Place, Boston, MA 02215. E-mail: george.king{at}joslin.harvard.edu.

Received for publication 26 October 2001 and accepted in revised form 10 June 2002.

CTGF, connective tissue growth factor; ECM, extracellular matrix; DAG, diacylglycerol; PKC, protein kinase C; STZ, streptozotocin; TBP, TATA binding protein; TGFß, transforming growth factor ß.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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